Hyaluronic acid and chondroitin sulfate based hydrolyzed collagen type II and method of making same

ABSTRACT

Hydrolyzed collagen type II powder compositions for inducing cartilage formation in an individual, method of preparing the compositions and use of the compositions in treating connective tissue disorder, replenishing skin viscoelasticity. The compositions are administered through an orally ingestible delivery medium for absorption into the gastrointestinal tract. The compositions are administered through a topical delivery medium for absorption into a dermis of the individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] (Not Applicable)

STATEMENT RE: FEDERALLY SPONSORED RESEARCH/DEVELOPMENT

[0002] (Not Applicable)

BACKGROUND OF THE INVENTION

[0003] The present invention relates to hydrolyzed collagen type IIpowder compositions for inducing cartilage formation in an individual, amethod of preparing the compositions and the use of the compositions intreating connective tissue disorder and replenishing skinviscoelasticity.

[0004] Studies have shown that collagen is a complex structural proteinwhich provides strength and flexibility to skin, hair and nails.Collagen is an essential and major component of muscles, tendons,cartilage, ligaments, joints and blood vessels in the human body. Thereare three main types of collagen: I, II and III. Types I and III areprimarily found in skin, tendon and bone. In contrast, type II is foundpredominantly in articular cartilage. Collagen is an unusual protein, inthat the proportion of glycine residues is nearly one-third, which isunusually high in comparison to other typical proteins. Proline is alsopresent to a much greater extent in collagen than in most otherproteins. Moreover, collagen contains two amino acids, 4-hydroxyprolineand 5-hydroxylysine, that are found in very few other proteins. Theamino acid sequence of collagen is remarkably regular, nearly everythird amino acid being glycine. In addition, the sequenceglycine-proline-hydroxyproline recurs frequently. In contrast, globularproteins rarely exhibit regularities in their amino acid sequences(Stryer, L., Biochemistry, Third Edition, W. H. Freeman and Co., NewYork, 1988, pp. 262).

[0005] In 1986, collagen was sold for the first time in the UnitedStates for use as a food supplement. Collagen (a mixture of Types I andIII) was extracted from calf skin tissue, hydrolyzed and prepared inpowdered form for use as a dietary supplement. The composition was soldunder the name “Hydrolyzed Collagen Beauty Supplement™” (Smarter Nails &Hair, Inc., Newport Beach, Calif.). In 1987, “Hydrolyzed Collagen BeautySupplement Tablet™” (Smarter Nails & Hair, Inc., Newport Beach, Calif.)was sold which comprised collagen powder and 10 mg vitamin C compressedinto 1,000 mg tablets.

[0006] U.S. Pat. No. 4,804,745 to Koepff et al. discloses agentscontaining collagen peptides produced by enzymatic hydrolysis for thetreatment of degenerative joint diseases. These peptides can be obtainedfrom animal skin, animal bones and other sufficiently purifiedconnective tissue and have average molecular weights of between 30 and45 kilodaltons.

[0007] U.S. Pat. No. 5,399,347 to Trentham et al. and Trentham et al.(Science 261:1727-1729, 1993) disclose the effective treatment ofrheumatoid arthritis (RA) with water-soluble whole chick collagen typeII or biologically active peptides derived therefrom. The mechanism bywhich the effect is believed to occur is via oral tolerization toautoantigens.

[0008] U.S. Pat. No. 5,364,845 to Henderson discloses a therapeuticcomposition and method for the protection, treatment and repair ofconnective tissue in mammals. This composition comprises glucosamine,chondroitin sulfate and manganese ascorbate. U.S. Pat. No. 5,587,363 toHenderson discloses a therapeutic composition and method for theprotection, treatment and repair of connective tissue in mammals whichincludes aminosugars and glycosaminoglycans.

[0009] Another well known substance which aids in the rejuvenation andtreatment of such ailments as connective tissue disorder is a substanceknown as Hyaluronic Acid (also known as “Hyaluronan” or “HA”) . It iswell known that the human body naturally contains such HA, as it isfound in several parts of the body such as the soft connective tissue,the vitreous body of the eye, hyaline cartilage, synovial joint fluid,the dermis, and the epidermis. Within these parts of the body, HA actsas a lubricant between connective tissues of the skin, protects thejoints by providing shock-absorption, and helps the body retain skinmoisture. Over time however, as the body ages, the amount of HA presentin the body deteriorates and the body may eventually develop one ofseveral health problems, in part due to a decreased presence of HA. Thiseffect is particularly apparent for those who are over the age of 50.Generally, the skin loses viscoelasticity, and wrinkles form ultimatelyas a result of this deficiency.

[0010] Due to the fact that HA is found in several different parts ofthe body, such a decrease in HA levels is associated with a greatvariety of disorders and ailments. For example, osteoarthritis patientsexperience decreased levels of HA in their synovial fluid. This has adetrimental effect on the joints because HA is primarily responsible forthe lubricating and shock-absorbing effects of the synovial fluid. Forthis reason, researchers have theorized that the replacement of suchlost HA may help osteoarthritis sufferers to rebuild damaged cartilageand to regain joint movement.

[0011] However, HA is not found exclusively in the human body. As amatter of fact, HA is found in most animals. For this reason, there hasbeen substantial research conducted to extract HA from other sources sothat humans may replenish the levels of HA that are lost over time. HAmay be extracted from several sources such as from micro-organisms(Streptococcal cultures) through a fermentation process or from theisolation HA from rooster combs. Of these major sources, rooster combsproduce HA having higher molecular weights. HA having a high molecularweight is too large to penetrate into the skin and the bloodstream andis therefore limited in its application. Generally, high molecularweight HA is used for eye surgery and cosmetics, specifically forcreating a viscoelastic film to retain skin moisture and to blockforeign substances.

[0012] For Osteoarthritis (“OA”) patients, one method of relieving jointpain is via a therapy known as viscosupplementation.Viscosupplementation requires an intra-articular injection of HA toalleviate the pain associated with OA and allows the patient to regainfunction of the affected joints. The end result of such treatment isthat the HA has an anti-inflammatory effect. Alternatively, the patientmay use intra-articular glucocorticoid injections. The benefit of bothinjections are somewhat comparable but the common disadvantage to boththerapies is the invasive nature of the injections.

[0013] Another constituent which alleviates pain in the joints and helpsrebuild connective tissue is an endogenous Glycosaminoglycan (“GAG”)called Chondroitin Sulfate (“CSA”) which is composed of Glucuronic Acid+N-acetyl-D-galactosamine+Glucosamine Sulfate. Generally, CSA is abiological polymer derived from connective tissue. There are three typesof Chondroitin Sulfate: Chondroitin Sulfate A (“CSA-A”) (shown in FIG.2) which is also known as chondroitin 6-sulphate, Chondroitin Sulfate B(“CSA-B”) (shown in FIG. 3) which is also known as dermatan sulphate,and Chondroitin Sulfate C (“CSA-C”) (shown in FIG. 4) which is alsoknown as chondroitin 4-sulphate. The ratio of CSA-A to CSA-C declines intandem with the progression of the aging process as does the content ofCSA-B in the Dermis of the skin. In this respect, CSA levels aregradually depleted and such a decrease in CSA levels has a similareffect to a decrease in HA levels in that the shock-absorption andlubrication qualities are reduced. Thus, like HA, CSA is generally animportant GAG for the maintenance of connective tissue.

[0014] Specifically, CSA-A has proven to be an effectiveanti-inflammatory that improves blood circulation, prevents IschemicHeart Disease, reduces incidences of heart attacks, reduces incidencesof strokes, and is also effective in supporting connective joint tissue.Insofar as Chondroitin Sulfate helps prevent heart disease, thisphenomenon is due to the inherent antithrombogenic or anticoagulantproperties which prevent abnormal blood clots and reduce the incidenceof strokes. CSA-B is found in the dermis of the skin and is also one ofthe constituents responsible for maintaining viscoelasticity of theskin. CSA-C inhibits Elastase, which is an enzyme that progressivelydegrades cartilage during the onset of Osteoarthritis.

[0015] Currently, Chondroitin Sulfate may be naturally ingested viaseafood. For example, mussels, oysters and shark cartilage naturallycontain Chondroitin Sulfate such that ingesting these types of foods mayhelp replenish lost Chondroitin Sulfate over time. However, it isimpractical to continually ingest seafood on a regular basis becausesome people are allergic to particular types of seafood, seafood isrelatively expensive, and some simply do not like the taste of seafood.Additionally, several Chondroitin Sulfate nutritional supplements arebeing sold currently for treating connective joint tissue. Most of thesesupplements derive the Chondroitin Sulfate from bovine cartilage orvelvet deer antler. In these forms, Chondroitin Sulfate is generally adifficult compound to digest in the gastrointestinal tract and the typeof Chondroitin Sulfate derived from such animals is generally not veryeffective in absorbing into the bloodstream.

[0016] There is a need for non-invasive therapies in treating OA andother connective tissue disorders which may be treated with HA and aneed for compositions capable of promoting repair of damaged connectivetissue. The present invention addresses this need.

BRIEF SUMMARY OF THE INVENTION

[0017] One embodiment of the present invention is hydrolyzed collagentype II, the hydrolyzed collagen having an average molecular weight ofbetween about 50 and 10,000 daltons. Preferably, the hydrolyzed collagentype II has an average molecular weight of about 5,500 daltons. In oneaspect of this preferred embodiment, the collagen is obtained fromchicken sternal cartilage. The hydrolyzed collagen type II preferablyincludes at least 20% depolymerized chondroitin sulphate and at least10% hyaluronic acid. The collagen may be formed into an orallyingestible delivery medium or a topical delivery medium. The collagen isa low molecular weight, thereby allowing the orally ingestible deliverymedium to readily absorb into the gastrointestinal tract of anindividual while the topical delivery medium readily absorbs into thedermis of an individual.

[0018] The present invention also provides a method of inducingcartilage formation in an individual with a connective tissue disorder,comprising orally administering to the individual an effective dailycartilage-inducing amount of hydrolyzed collagen type II. The connectivetissue disorder includes degenerative joint diseases, joint defects,osteoarthritis, polychondritis, vascular disease and cartilage injuries.Preferably, the effective daily amount is between about 500 and 5,000mg. More preferably, the effective daily amount is between about 1,000and 4,000 mg. Most preferably, the effective daily amount is betweenabout 2,000 and 3,000 mg.

[0019] Another embodiment of the invention is a method of replenishinghyaluronic acid and chondroitin sulfate, restoring skin viscoelasticity,retaining skin moisture, healing wounds and improving the overallappearance of skin comprising topically administering to an individual,or other animal, a selectable amount of chicken sternalcartilage-derived material comprising hydrolyzed collagen type II havingan average molecular weight of between about 50 and about 10,000daltons. In this embodiment, the patient is given the option of applyingas much or as little as desired to portions of the body as theindividual sees fit (i.e., the eyes).

[0020] Still another embodiment of the invention is a method ofpreparing hydrolyzed collagen type II powder, comprising the followingsteps: cutting fresh chicken sternal cartilage to within not less thanabout 2 mm of the bone; suspending the cartilage in an aqueous solution;treating said cartilage with a proteolytic enzyme to form a hydrolysate,said proteolytic enzyme being capable of hydrolyzing collagen type II tofragments having an average molecular weight of between 50 to 10,000daltons; sterilizing the hydrolysate; filtering the hydrolysate;concentrating the hydrolysate; and drying the hydrolysate to form acollagen type II powder. The method may further comprise the step offreezing the cartilage after the cutting step. Preferably, the aqueoussolution is water. Advantageously, the enzyme is papain, ficin orbromelain. In one aspect of this preferred embodiment, the sterilizingstep comprises heating the hydrolysate at 95° C. for about 30 minutes.Preferably, the drying step comprises spray drying. Preferably, the pHof the suspending and treating steps is between about 4 and 8.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a schematic diagram of the process for preparing thehydrolyzed collagen type II powder of the invention.

[0022]FIG. 2 is a molecular diagram showing Chondroitin Sulphate-A.

[0023]FIG. 3 is a molecular diagram showing Chondroitin Sulphate-B.

[0024]FIG. 4 is a molecular diagram showing Chondroitin Sulphate-C.

[0025]FIG. 5 is a molecular diagram showing Hyaluronic Acid.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides a hydrolyzed, denatured collagentype II composition, method for preparing the composition and use of thecomposition in the prevention, treatment and repair of cartilagedefects. The method involves cutting fresh sternal cartilage fromchicken carcasses and removing all meat therefrom. The sternal cartilageis cut, leaving a space of about two millimeters from the bone so as tonot remove any bone fragments. This is critical to the purity of thefinal product because it avoids contamination of collagen type II withtypes I and III found in bone. Collagen type II contains the greatestconcentration of proteoglycans, which help rejuvenate connective tissuejoints. Therefore, maintaining a pure collagen type II compound ensuresthat the highest concentration of such proteoglycans. The fresh sternalcartilage is then promptly frozen and the remains of the chicken carcassare discarded. It is exclusively the sternal cartilage that is used forpreparing the collagen type II powder. The chicken sternal cartilage isprocessed according to good manufacturing procedures (GMP).

[0027] Other contemplated sources of collagen type II are mammaliancartilage (e.g. bovine, porcine and avian) and shark fins. However, suchforms of collagen type II are generally less effective in absorbing intothe gastrointestinal tract. On the other hand, the present inventionnaturally contains depolymerized chondroitin sulfate. This particularform of chondroitin sulfate derived from the chicken sternal cartilageabsorbs into the gastrointestinal tract approximately three to fivetimes better than traditional bovine derived forms which may not bedepolymerized. In this respect, the depolymerized chondroitin sulfate isbroken down into its individual monomers for an increased ability toabsorb into the gastrointestinal tract, thereby increasing the bloodconcentrations of glycosaminoglycans rich in hydrolyzed collagen typeII. Preferably, the present invention includes 20-25% depolymerizedchondroitin sulfate, a high concentration of this easily absorbableconstituent. However, this depolymerized chondroitin sulfate alsoprovides for absorption into the body via other means. For example, thedepolymerized chondroitin sulfate in the present invention may absorbinto the skin when applied topically to replenish CSA-C in the dermis.

[0028] Similarly, the hyaluronic acid present in the collagenreplenishes lost levels of such hyaluronic acid in the body bysupplementing the synovial joint fluid, cartilage, eyes, skin tissue,connective tissue, nails, and other parts of the body which graduallylose hyaluronic acid over time. The collagen having the hyaluronic acidalso provides structural support to joints, promotes new cartilagesynthesis, helps heal wounds and is useful in a myriad of otherapplications where hyaluronic acid and chondroitin sulfate is generallyfound within the human body, or animal body. Advantageously, because thecollagen is a low molecular weight, the hyaluronic acid contained in thecollagen is capable of absorbing into the gastrointestinal tract viaoral delivery mediums and can absorb into the skin when appliedtopically. Thus, hyaluronic acid is also capable of reaching the coriumlayers of the dermis via oral ingestion. In this respect, the hydrolyzedcollagen type II and its unique low molecular weight properties allowfor the hyaluronic acid, chondroitin sulfate, glucosamine sulfate,cartilage matrix glycoprotein (CMGP), and other glucosaminoglycans toall absorb into the gastrointestinal tract via oral delivery mediums andinto the skin via topical delivery mediums.

[0029] The production of hydrolyzed collagen type II in powdered form isshown in FIG. 1. Whole cartilage is suspended in an aqueous solution,preferably water for about one hour at about 35° C. at a pH of betweenabout 4 and 8. In a preferred embodiment, the pH is between about 6 and7. In a more preferred embodiment, the pH is about 6.5. The water isremoved, and the cartilage is incubated with one or more proteasesobtainable from a natural source (i.e. papain, ficin, bromelain) forbetween about 2 and 10 hours, preferably about 6 hours, at about 35°C.-55° C. at a pH of between about 4 and 8 to form a hydrolysate. The pHwill depend on the pH optimum of the particular enzyme(s) used for thehydrolysis and are well known to one of ordinary skill in the art. Thehydrolysate is then sterilized for about 30 minutes at a temperaturebetween about 95° C. and 105° C. The sterilized hydrolysate is filteredthrough diatomaceous earth, concentrated, preferably under vacuum, driedto form a powder and packed. Other filtration methods are contemplated,including vacuum filtration. In a preferred embodiment, the hydrolyzedcollagen type II is spray dried using a size 56 pressure nozzle into aheat tunnel. The final particle size and mesh are adjusted to 0.46 g/cc,yielding a fine powder. The powder is packed in a 40 kg drum with aplastic bag liner. The powder is water soluble.

[0030] The average molecular weight of the final product is between 50and 10,000 daltons, preferably 5,500 daltons. The moisture content isbetween 5% and 7%. The final product is high in mucopolysaccharides,particularly chondroitin sulfate and glucosamine sulfate. Suchmucopolysaccharaides are major constituents in providingshock-absorption and lubrication in the connective tissue joints. Theproduct has 375 calories per 100 grams, contains 67% protein (12.1%total nitrogen), 18% carbohydrate and 0.1% fat. The amino acidcomposition of the hydrolysate differs substantially from typicalcollagens and is shown in Table 1. Hydroxyproline is low, hydroxylysineis absent and tryptophan is low. The low molecular weight and high aminoacid composition promote optimal assimilation of the peptides. TABLE 1Amino acid composition of hydrolyzed collagen type II Amino acid g/100 gproduct arginine 4.42 histidine 2.05 isoleucine 1.90 leucine 4.20 lysine3.54 methionine 1.38 phenylalanine 2.14 threonine 2.60 tryptophan 0.37alanine 4.51 asparagine/aspartic acid 5.29 cystine 0.46glutamine/glutamic acid 8.75 glycine 8.93 hydroxyproline 3.90 proline5.25 serine 2.45 tyrosine 1.16 valine 2.43

[0031] A test analysis of the hydrolyzed collagen yields results whichindicate that at least 20% of depolymerized chondroitin sulfate ispresent in the collagen. Additionally, at least 10% of hyaluronic acidis also found therein. However, because the collagen is extracted from anatural source (i.e. chicken sternum), the actual percentage ofdepolymerized chondroitin sulfate and hyaluronic acid may vary. Thehighest concentration of depolymerized chondroitin sulfate andhyaluronic acid obtainable is preferred. TABLE 2 Test Results ofHydrolyzed Collagen Type II Specifications Typical analysis Purity 100%Solubility Water-Soluble Chondroitin Sulfate ˜20.0% Hyaluronic Acid˜10.0% Other Proteoglycans ˜3.0% Collagen Type II Protein ˜62.7%Nitrogen 12.1% Fat 0.0% Loss on Drying <8% Heavy Metals <5 ppm Ash 8.3%Calcium 530 mg/100 gms Magnesium 161 mg/100 gms Potassium 1961 mg/100gms Microbiological Analysis Standard Plate Count <1000 gms E. ColiNegative Salmonella Negative Physical Analysis Appearance Offwhite/yellow fine powder Odor Characteristic Particle Size >100 mesh

[0032] When taken orally by an individual with a connective tissuedisorder, hydrolyzed collagen type II helps build cartilage andsignificantly improves the disorder. The lost levels of HA, CSA-A,CSA-B, and CSA-C in the joints are replenished via ingestion into thegastrointestinal tract. Furthermore, oral ingestion of the hydrolyzedcollagen type II helps contribute to the wound healing process due tothe chondroprotective nature of the collagen's composition.

[0033] “Oral” administration includes oral, enteral or intragastricadministration. The hydrolyzed collagen type II of the invention can beused to treat, for example, degenerative joint diseases (i.e. rheumatoidarthritis), joint defects, osteoarthritis, polychondritis, vasculardisease, cartilage injuries, silicone poisoning due to ruptured breastimplants, autoimmune diseases involving connective tissue autoantibodies(i.e. rheumatoid arthritis), progressive myopia, Menier's disease andany other connective tissue disorder which would benefit from increasedsynthesis of cartilage. The hydrolyzed collagen type II alsosignificantly reduces sun-induced skin wrinkles.

[0034] By extracting HA from chicken sternal cartilage, a low molecularweight HA may be obtained by hydrolysis after such extraction. Thisdistinction is crucial because the beneficial therapeutic activity of HAis mostly dependent upon the molecular weight of HA. Due to the lowmolecular weight of the HA found in the hydrolyzed collagen type II, thehydrolyzed collagen type II readily absorbs into the gastrointestinaltract and allows the rejuvenating constituents of HA and CSA to restoreviscoelasticity to the skin, protect connective tissues, promotecartilage synthesis, retain skin moisture, heal wounds and improve theoverall appearance of skin. In its oral form, the hydrolyzed collagentype II is capable of reaching the corium layers of the skin whichtypically cannot be reached by topical applications.

[0035] For oral administration as a nutritional supplement, therapeuticor prophylactic agent, the hydrolyzed collagen type II of the inventionmay be provided as a dispersible powder or granule, tablet, aqueous oroil suspension, emulsion, hard or soft capsule, syrup or elixir.Advantageously, the ability to provide the hydrolyzed collagen type IIin such a variety of forms allows the invention to be used by itself orin conjunction with several different applications.

[0036] The low molecular weight of the hydrolyzed collagen type IIallows the powder form to be integrated into topical gels. In thisrespect, the gel may be applied directly to the skin and ultimately, thecollagen readily absorbs into the skin. Absorption directly into theskin provides the advantage of moisturizing the skin, maintaining fluidbalance in the cells of the skin and providing a thin layer ofviscoelastic protective film which keeps foreign substances fromattacking the skin. Additionally, the film retains water and moisturizesthe skin in much the same way that hyaluronic acid does in theintercellular matrix of dermal connective tissue. Ultimately suchapplication helps the skin appear younger, reduces the effects of agingupon the skin, and accelerates the wound healing process when thecollagen is applied to wounds. Additionally, topical applicationprovides the advantage of a targeted solution to where the consumerneeds it the most. For example, the topical collagen user's predominatepurpose may be to rejuvenate the skin and restore viscoelasticity toimprove the overall appearance of the skin. Such a user may then applythe topical collagen to the desired parts of the body, such as under theeyes, to concentrate on rejuvenating that particular area. A topicalform may also be used for administering the collagen to wounds forreducing the appearance of scarring and accelerating the overall woundhealing process. On the other hand, an orally ingestible form providesthe advantage of allowing a total body rejuvenation wherein the collagenrestores hyaluronic acid and chondroitin sulfate constituents to partsof the body which are deficient in these chemicals.

[0037] As a cosmetic, the collagen may be integrated with existingfacial cosmetics, such as foundation, concealer, and lipstick as anadditive which works invisibly to replenish the lost constituents. Inthis respect, the cosmetic user may apply the daily cosmetics as usualwithout the requirement of an additional moisturizer due to thecollagen's ability to help retain natural skin moisture. Furthermore, itis contemplated that the collagen may be used in conjunction with othertypical consumer products where skin moisturizing is also useful. Forexample, some soaps include a moisturizer as an ingredient to counteractany drying effects detergents may have on the skin. The collagen may beused with a moisturizer to also counteract such effects whilesimultaneously replenishing lost levels of HA and CSA.

[0038] The hydrolyzed collagen type II may serve as an ingredient of aproduct medication manufactured to facilitate healing of wounds andinjuries. In this regard, such product medication, which incorporatesthe hydrolyzed collagen type II as one of its ingredients, may bedirectly medicated on areas of skin where the wound occurs.Additionally, the collagen may be integrated into an orally ingestibleform of wound healing medication by absorbing into the gastrointestinaltract. Of course, sufficient quantity should be applied to promote ahealing process of the wound.

[0039] Specifically, the hydrolyzed collagen type II of the presentinvention may be conformed to provide topical applications so as toaccelerate healing of wounds and injuries associated with thediseases/disorders described above (e.g., cartilage injuries, hepatitis,decubititis). In this respect, when a wound heals, the fibroplastssecrete glycosaminoglycans such that a hydrophilic matrix is formed. Thehydrophilic property of the hydrolyzed collagen may then provide agreater degree of epithelialization. The increased presence of suchglycosaminoglycans assists the fibroblasts which are continually movingand replicating during the wound healing process. Thus, the overallprocess for accelerating the healing of cutaneous wounds is acceleratedby applying the collagen to the wound. In the same respect, the sameproperties of the collagen assist in plastic surgery applications forburn victims. Applying the collagen to wounds, skin graft sites, andskin donor sites may reduce inflammation, reduce the incidence ofscarring, and also increase the overall healing process.

[0040] Compositions intended for oral use may be prepared according toany method known in the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more of thefollowing agents: sweeteners, flavoring agents, coloring agents andpreservatives. The sweetening and flavoring agents will increase thepalatability of preparation. Tablets containing the hydrolyzed collagentype II in admixture with non-toxic pharmaceutically acceptableexcipients suitable for tablet manufacture are acceptable. Suchexcipients include inert diluents such as calcium carbonate, sodiumcarbonate, lactose, calcium phosphate or sodium phosphate; granulatingand disintegrating agents, such as corn starch or alginic acid; bindingagents such as starch, gelatin or acacia; and lubricating agents such asmagnesium stearate, stearic acid or talc. Tablets may be uncoated or maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period of time. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed. The use of enteric coatings is also contemplated.

[0041] Formulations for oral use may also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example calcium carbonate, calcium phosphate or koalin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, such as peanut oil, liquid paraffin or oliveoil.

[0042] Aqueous suspensions may contain the hydrolyzed collage type II ofthe invention in admixture with excipients suitable for the manufactureof aqueous suspensions. Such excipients include suspending agents,dispersing or wetting agents, one or more preservatives, one or morecoloring agents, one or more flavoring agents and one or more sweeteningagents such as sucrose or saccharin.

[0043] Oil suspensions may be formulated by suspending the activeingredient in a vegetable oil, such as arachis oil, olive oil, sesameoil or coconut oil, or in a mineral oil such as liquid paraffin. The oilsuspension may contain a thickening agent, such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents, such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by an added antioxidantsuch as ascorbic acid. Dispersible powders and granules of the inventionsuitable for preparation of an aqueous suspension by the addition ofwater provide the active ingredient in admixture with a dispersing orwetting agent, a suspending agent, and one or more preservatives.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

[0044] Syrups and elixirs may be formulated with sweetening agents, suchas glycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring agent and/or a coloring agent.

[0045] The hydrolyzed collagen type II powder may be mixed with otheringestible forms and consumed in solid, semi-solid solution, suspensionor emulsion form. It may also be mixed in conjunction or alternativelywith pharmaceutically acceptable carriers, flavor enhancers, water,suspending agents and emulsifying agents. In a preferred embodiment, thehydrolyzed collagen type II powder is mixed with a citrus juice such asorange, grapefruit or tangerine due to the promotion of connectivetissue formation by ascorbic acid. The hydrolyzed collagen may also beprovided in tablet form in admixture with ascorbic acid.

[0046] For use as a nutritional supplement, prophylactic or therapeuticagent, the hydrolyzed collagen is orally administered in a daily dosageof between about 500 mg and 5,000 mg. More preferably, it isadministered in a daily dosage of between about 2,000 mg and 4,000 mg.Most preferably, it is administered in a daily dosage of between about2,000 and 3,000 mg per day. The hydrolyzed collagen type II powder maybe formulated into tablets which range from 300 mg to 1,000 mg pertablet. In a preferred embodiment, the hydrolyzed collage type II powderis formulated into 500 mg tablets and 4-6 tablets are taken daily. Inanother preferred embodiment, the tablets are taken on an empty stomachwith a beverage containing vitamin C.

[0047] In another preferred embodiment, the hydrolyzed collagen type IIpowder is mixed with water or a citrus juice prior to ingestion. Thepreparations described above can be taken indefinitely by individualsaffected by connective tissue disorders or by healthy individuals as apreventative agent. If desired, an individual with such a disorder cantake the preparation until no further improvement is noted in thedisorder.

[0048] It is contemplated that application of the medication may beaccomplished by wiping using sterile gloves, applying with appropriatedevices, spray, painting, or other means to produce a continuousadhesive film on the wound. If additional support for the wound isrequired, bandages, splints, other medication dispensing devices,prosthetic devices or any other devices may be applied in conjunctiontherewith.

[0049] In addition, the hydrolyzed collagen type II of the presentinvention may further be adhered as a preventive remedy. For instance,like many other ingredients in the marketplace, the hydrolyzed collagentype II can be subjected to cosmetic applications used for anti-agingpurposes. More particularly, as the hydrolyzed collagen type II can helpreduce sun-induced skin wrinkles, it may therefore be integrated into ananti-aging cosmetic as one of its essential ingredients. As such, thehydrolyzed collagen type II of the present invention can be utilized ina variety of exemplary manners due to its primary inherentcharacteristic of inducing cartilage formation.

[0050] The above detailed description of the invention is set forthsolely to assist in understanding the invention. It is to be understoodthat variations of the invention, including all equivalents now known orlater developed, are to be considered as falling within the scope of theinvention, which is limited only by the following claims.

1. A method for preparing chicken sternal cartilage-derived materialcomprising hydrolyzed collagen type II, comprising the following steps:(a) cutting fresh chicken sternal cartilage to within not less thanabout 2 mm of the bone; (b) suspending said cartilage in an aqueoussolution; (c) treating said cartilage with a proteolytic enzyme to forma hydrolysate, said proteolytic enzyme being capable of hydrolyzingcollagen type II to fragments having an average molecular weight ofbetween about 50 to about 10,000 daltons; (d) sterilizing saidhydrolysate; (e) filtering said hydrolysate; (f) concentrating saidhydrolysate; (g) drying said hydrolysate to form a powder enriched incollagen type II powder; and (h) isolating said powder enriched incollagen type II.
 2. The method of claim 1, further comprising the stepof freezing said cartilage after said cutting step.
 3. The method ofclaim 1, wherein said aqueous solution is water.
 4. The method of claim1, wherein said enzyme is selected from the group consisting of papain,ficin and bromelain.
 5. The method of claim 1, wherein said sterilizingstep comprises heating said hydrolysate at about 95° C.-105° C. forabout 30 minutes.
 6. The method of claim 1, wherein said drying stepcomprises spray drying.
 7. The method of claim 1, wherein step (b) isperformed at a pH between 4 and
 8. 8. The method of claim 1, whereinstep (c) is performed at a pH between 4 and
 8. 9. The method of claim 1,wherein said material comprises depolymerized chondroitin sulfate. 10.The method of claim 9, wherein said depolymerized chondroitin sulfate isat least 20% of said material.
 11. The method of claim 1, wherein saidpowder comprises hyaluronic acid.
 12. The method of claim 11, whereinsaid hyaluronic acid is at least 10% of said material.
 13. Hydrolyzedcollagen type II for inducing cartilage formation in an individual, saidhydrolyzed collagen being derived from chicken sternal cartilage, saidhydrolyzed collagen having an average molecular weight of between about50 and about 10,000 daltons.
 14. The hydrolyzed collagen type II ofclaim 13, wherein said hydrolyzed collagen is formed into an orallyingestible delivery medium.
 15. The hydrolyzed collagen type II of claim14, wherein said orally ingestible delivery medium is chosen from thegroup consisting of powder, tablet, aqueous suspension, emulsion,capsule, elixir and combinations thereof.
 16. The hydrolyzed collagentype II of claim 13, wherein said hydrolyzed collagen is formed into atopical delivery medium.
 17. The hydrolyzed collagen type II of claim16, wherein said topical delivery medium is a topical gel absorbableinto a dermis.
 18. The hydrolyzed collagen type II of claim 13, whereinsaid hydrolyzed collagen is derived from a mammalian cartilage.
 19. Thehydrolyzed collagen type II of claim 13, wherein said hydrolyzedcollagen is derived from a shark fin.
 20. The hydrolyzed collagen typeII of claim 13, wherein said hydrolyzed collagen comprises depolymerizedchondroitin sulfate.
 21. The hydrolyzed collagen type II of claim 20,wherein said depolymerized chondroitin sulfate is at least 20% of saidhydrolyzed collagen.
 22. The hydrolyzed collagen type II of claim 13,wherein said hydrolyzed collagen comprises hyaluronic acid.
 23. Thehydrolyzed collagen type II of claim 22, wherein said hyaluronic acid isat least 10% of said hydrolyzed collagen.
 24. A method for treatingconnective tissue disorder comprising orally administering an effectivedaily amount of chicken sternal cartilage-derived material comprisinghydrolyzed collagen type II having an average molecular weight ofbetween about 50 and about 10,000 daltons.
 25. The method of claim 24,wherein said hydrolyzed collagen comprises depolymerized chondroitinsulfate.
 26. The method of claim 25, wherein said depolymerizedchondroitin sulfate is at least 20% of said hydrolyzed collagen.
 27. Themethod of claim 24, wherein said hydrolyzed collagen compriseshyaluronic acid.
 28. The method of claim 27, wherein said hyaluronicacid is at least 10% of said hydrolyzed collagen.
 29. A method ofreplenishing hyaluronic acid and chondroitin sulfate comprisingtopically administering a selectable amount of chicken sternalcartilage-derived material to a dermis, said material comprisinghydrolyzed collagen type II having an average molecular weight ofbetween about 50 and about 10,000 daltons.
 30. The method of claim 29,wherein said hydrolyzed collagen comprises depolymerized chondroitinsulfate.
 31. The method of claim 30, wherein said depolymerizedchondroitin sulfate is at least 20% of said hydrolyzed collagen.
 32. Themethod of claim 29, wherein said hydrolyzed collagen compriseshyaluronic acid.
 33. The method of claim 32, wherein said hyaluronicacid is at least 10% of said hydrolyzed collagen.